Inhibitory Antibodies to Human Angiotensin-Converting Enzyme:  Fine Epitope Mapping and Mechanism of Action

Abstract

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.

A further case of renal tubular dysgenesis surviving the neonatal period

“…The first of these two deletions destabilizes the mRNA. The latter, which consists of an alpha-helix structure [6,8] and conserved residues in mouse, presumably leads to significant reductions in the serum ACE concentration, thereby causing the RTD.…”

Functional single-cell hybridoma screening using droplet-based microfluidics

“…Heterogeneous hybridoma cell populations [4E3 and Elec-403 (13)(14)(15)18) cells in a ratio of 1∶75; 1∶1;000 or 1∶10; 000] were encapsulated into 660 pL drops at a density of 1.25 × 10 6 cells∕mL together with 1.6 ng∕mL ACE-1 (R&D Systems). The resulting emulsion was incubated off-chip for 6 h at 37°C under a 5% CO 2 atmosphere, followed by reinjection into the integrated microfluidic chip (Fig.…”

“…However, the two catalytic sites have different substrate specificities and catalytic properties as well as different affinities for competitive inhibitors (12,13). Monoclonal antibodies against this enzyme have been used in structural and functional studies of ACE-1 (14,15) and mAbs against ACE-1 have both diagnostic and therapeutic potential: ACE-1 is a target for drugs to treat hypertension and congestive heart failure (16). Here we demonstrate the enrichment of hybridoma cells secreting the ACE-1 inhibitory mAb 4E3 (14,15) from a large excess of unrelated hybridoma cells.…”

Immunotargeting of the Pulmonary Endothelium via Angiotensin-Converting-Enzyme in Isolated Ventilated and Perfused Human Lung

“…Three different mouse anti-human ACE mAbs recognizing different epitopes on the N domain of human ACE were compared in this study for their ability to accumulate in the human lung during warm lung perfusion -9B9 (Danilov et al 1994Balyasnikova et al 2002b ;Gordon et al 2010 ) , i2H5 (Balyasnikova et al 2002b ;Danilov et al 1994 ;Skirgello et al 2006 ) and 3G8 (Danilov et al 1994 ;Gordon et al 2010 ) .…”